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Journal of Advanced Laboratory Research in Biology
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e-ISSN 0976-7614
Volume 4, Issue 1, January 2013
Research Article
Short-term exposure to quinalphos induced biochemical and haematological changes in
freshwater fish, Oreochromis mossambicus
K.C. Chitra*, P. Nikhila and K.P. Asifa
*Department of Zoology, University of Calicut, Malappuram–673635, Kerala, India.
Abstract: The toxic impact of quinalphos, an organothiophosphate, on the biochemical as well as haematological
parameters was studied in the adult freshwater fish, Oreochromis mossambicus. In the present study, 0.5µl/ L
quinalphos was chosen to represent sublethal concentration for 48 and 96 hours as short-term exposure and
respective control animals were maintained. Quinalphos induced toxic stress to the exposed fishes, which is obvious
by the reduction in the oxygen consumption of the fishes at the time of exposure and this could be due to shrinkage
of the respiratory epithelium or possibly due to mucus accumulation on gills. Decrease in the haemoglobin content
was observed and this may be due to either an increase in the rate at which the haemoglobin is destroyed or
decreased rate of haemopoietic potential of the fish. In the present study, the significant increase in WBC count
indicates hypersensitivity of leucocytes to quinalphos and these changes may be due to immunological reactions to
produce antibodies to cope up with the stress. Decrease in the level of RBC count indicated decrease in
erythropoietic activity or severe anemic state. Reduction in the plasma and tissue protein of quinalphos exposed
fishes may be due to its utilization to mitigate the energy demand when the fishes are under stress. Increase of
plasma glucose and the total glucose content in tissues like liver, muscle and gill might have resulted from
gluconeogenesis to provide energy for the increased metabolic demands imposed by quinalphos stress. Thus the
biochemical and hematological alterations due to acute short-term exposure to quinalphos were due to the toxic
stress of the toxicant.
Keywords: Quinalphos, Haemoglobin, Fish, Oxygen Consumption, Glucose.
1.
Introduction
The use of insecticides are being increased in the
recent years to control the pest, in which only 1% of the
pesticide applied hits the target pest while, the
remaining 99% of the pesticide drifts into the
environment contaminating soil, water and biota. This
poses a constant threat to the non-target organisms
especially to fishes; because pesticides are known to
alter their behavior pattern, growth, nutritional value
and physiology. Therefore, it becomes a matter of great
concern when aquatic pollution due to pesticides are
discussed. Quinalphos is one of the organophosphate
insecticides extensively used in agriculture, which has
become a matter of concern today because of its
potentiality and hazardous effect. The recommended
dose of quinalphos for field application is 0.188mg of
*Corresponding author:
E-mail: kcchitra@yahoo.com; Tel: +919495135330.
quinalphos per liter of water on rice field. Among this
50% of concentration goes into the water where fish is
cultivated and ultimately lead to deleterious effects on
aquatic organisms.
One of the early symptoms of acute pesticide
poisoning is failure of respiratory metabolism where
some pesticides decrease the oxygen uptake of fish. The
rate of oxygen consumption can be used as a biodetector in monitoring the physiological effects of
toxicants and the oxygen consumption pattern will
indicate the possible mapping of metabolic pathways
influenced by the toxicant stress (Mushigeri and David,
2002). Apart from this haemolysis of red blood cells
provides simple and rapid way of understanding the
effect of pollutants on biological membranes
(Harington et al., 1971).
Short-term exposure to quinalphos in Oreochromis mossambicus
Blood is the medium of intercellular and
intracellular transport, which comes in direct contact
with various organs and tissues of the body, the
physiological state of an animal at a particular time is
reflected in its blood. Thus blood parameters are
considered as a pathophysiological indicator of the
entire body and therefore important in diagnosing the
structural and functional status of fishes exposed to
toxicants (Sarkar et al., 2004; Maheswaran et al.,
2008). So fish blood is being studied increasingly in
toxicological research and environmental monitoring as
a possible indicator of physiological and pathological
changes in fishery management and disease
investigations. Fish live in very intimate contact with
their environment and are therefore very susceptible to
physical and chemical changes which may be reflected
in their blood components. Alteration in haematological
and biochemical parameters of toxicant treated fish has
recently emerged as an important tool for water quality
assessment in the field of environmental toxicology.
These studies could be used to indicate the health status
of fish as well as water quality. Thus the objective of
the present investigation is to determine the short-term
effect of quinalphos on some selected haematological
and biochemical parameters in freshwater fish,
Oreochromis mossambicus and related effect from this
exposure as a way to evaluate the toxicity risk of
quinalphos to the test species.
2.
Material and methods
2.1 Chemicals
Quinalphos
(O,O-diethyl
O-quinoxalin-2-yl
phosphorothioate; 70% EC), was a generous gift from
Hikal Laboratory, Gujarat, India. All other chemicals
were of analytical grade and obtained from local
commercial sources.
2.2 Treatment
The lethal concentration for 50% killing of
animals, (LC50) values was computed on the basis of
probit analysis (Finney, 1971) for 96 h, which was
5µl/L (Chitra et al., 2012). In the present study, onetenth of the dosage (0.5µl/L) quinalphos was chosen to
represent sublethal concentration for 48 and 96 hours as
short-term exposure and respective control animals
were also maintained. Fishes of control and treatment
groups were continuously monitored for acute toxicity
testing. Oxygen consumption of fishes was measured to
test the energy metabolism of the test animal. The body
weights of fishes as well as the weights of organs as gill
and liver from control and treatment groups were also
measured at the end of the treatment. Biochemical and
haematological examinations was performed at the end
of each treatment.
2.3 Measurement of oxygen consumption
Oxygen consumption of fish was measured using
modified Winkler’s method (Kunnemann and
J. Adv. Lab. Res. Biol.
Chitra et al
Bashamohideen, 1978). To the collected sample of
water in a 300ml BOD bottle 2ml MnSO4 solution was
added and followed by 2ml alkaline-iodide-azide
reagent. The bottle was stopper carefully to exclude air
bubbles and mixed by inverting bottle a few times.
When precipitate has settled completely 2ml
concentrated H2SO4 was added, recapped and mixed by
inverting several times until the solution was
completely mixed, then it was titrated against sodium
thiosulphate solution. When it becomes light yellow, 2
or 3 drops of starch was added till it reaches colorless
endpoint. The oxygen consumption of fish was then
expressed in ml of oxygen consumed/g wet wt. of
fish/h.
2.4 Tissue processing and biochemical analysis
At the end of each exposure period, fishes were
caught very gently using a small dip net, one at a time
with least disturbance. Each fish was held and wrapped
with a clean, dry filter paper and the posterior half of its
body was blotted with another clean coarse filter paper.
Caudal peduncle of the fish (control and experimental
group) was severed at a single stroke using a sharp
blade. After discarding the first drop of blood, the
freely oozing blood was collected in a small watch
glass containing a sufficient quantity of anticoagulant
(2% ethylenediaminetetraacetic acid - EDTA). The
blood was thoroughly mixed with the anticoagulant
using a thin, blunt glass rod, during the process of
collection itself.
The whole blood was used for the estimation of
erythrocyte and leucocytes counts (Rusia and Sood,
1992) and haemoglobin (Drabkin, 1946) in the control
and experimental groups. The remainder of the blood
sample was centrifuged at 4000 rpm for 3 minutes to
separate the plasma, which was then used for the
biochemical estimation of plasma protein (Lowry et al.,
1951) and carbohydrate (Trinder, 1969). Tissues such
as gill, liver and muscle from control and treatment
groups were also removed to analyze the total protein
and carbohydrate.
2.5 Statistical analyses
Statistical analyses were performed using one-way
analysis of variance (ANOVA) and the means were
compared by Duncan’s Multiple Range test using
statistical package SPSS 17.0. Differences were
considered to be significant at p<0.05 against control
groups. Data are presented as mean  SD for ten
animals per group. All biochemical estimations were
carried out in duplicate.
3.
Results
3.1 Effect of quinalphos on body weights
Exposure to quinalphos at the dose of 0.5µl/L for
96 h showed a significant decrease in the body weight.
However, no significant changes in the body weights
were observed in 48 h exposed fishes (Fig. 1).
2
Short-term exposure to quinalphos in Oreochromis mossambicus
Chitra et al
mainly on the dorsal part. The surface of the body was
opaque with slightly increased amount of mucus and
with expressive pigmentation mainly on the dorsal part.
An excess secretion of mucous in fish forms a nonspecific response against toxicants, thereby probably
reducing toxicant contact. It also forms a barrier
between the body and the toxic medium, so as to
minimize its irritating effect, or to scavenge it through
epidermal mucus.
Fig. 1. Effect of quinalphos on the body weights of the fish,
Oreochromis mossambicus.
3.4 Effect of quinalphos on oxygen consumption
A reduction in oxygen consumption was observed
after the fish exposed to quinalphos at the dose of
0.5µl/L for 48 and 96 hours (Fig. 4).
3.2 Effect of quinalphos on organ weights
The weights of organs as liver and gill decreased
significantly at the end of 48 and 96 hours of
quinalphos exposure when compared with those of
control fishes (Figs. 2 and 3).
Fig. 4. Effect of quinalphos on the oxygen consumption of the
freshwater fish, Oreochromis mossambicus.
Fig. 2. Effect of quinalphos on the weights of liver of the fish,
Oreochromis mossambicus.
3.5 Effect of quinalphos on haematological
parameters
Quinalphos at the dose of 0.5µl/L for 48 and 96
hours showed a significant decrease in the level of Hb
(%) and total RBC whereas WBC count was increased
significantly (p<0.05) when compared to the control
animals (Figs. 5, 6 and 7).
Treatment of quinalphos at the dose of 0.5µl/L for
48 and 96 hours showed a significant (p<0.05) increase
in the level of blood glucose whereas it significantly
decreased the blood protein in the treated groups as
compared with the control fishes (Figs. 8 and 9).
Fig. 3. Effect of quinalphos on the weights of gill of the fish,
Oreochromis mossambicus.
3.3 Acute toxicity
In the toxic environment, fish exhibited irregular,
erratic, and darting swimming movements, and loss of
equilibrium followed by hanging vertically in water.
They slowly became lethargic, restless, and secreted
excess mucus all over the body. Body surface
darkening was noticeable in this phase of poisoning,
J. Adv. Lab. Res. Biol.
Fig. 5. Effect of quinalphos on haemoglobin level in the blood of the
fish, Oreochromis mossambicus.
3
Short-term exposure to quinalphos in Oreochromis mossambicus
Chitra et al
3.6 Effect of quinalphos on biochemical parameters
Exposure to quinalphos at the dose of 0.5µl/L for
48 and 96 hours showed a significant decrease in the
protein whereas increased the level of glucose in liver,
muscles and gills of the quinalphos-treated fishes than
that of the control group (Figs. 10 and 11).
Fig. 6. Effect of quinalphos on RBC count in the blood of the fish,
Oreochromis mossambicus.
Fig. 10. Effect of quinalphos on the tissue protein of the fish,
Oreochromis mossambicus.
Fig. 7. Effect of quinalphos on WBC count in the blood of the fish,
Oreochromis mossambicus.
Fig. 11. Effect of quinalphos on the level of glucose in the tissues of
the fish, Oreochromis mossambicus.
4.
Fig. 8. Effect of quinalphos on the random blood glucose in the
blood of the fish, Oreochromis mossambicus.
Fig. 9. Effect of quinalphos on total protein level in the blood of the
fish, Oreochromis mossambicus.
J. Adv. Lab. Res. Biol.
Discussion
Aquatic ecosystems that run through agricultural
areas have high probability of being contaminated by
run-off and groundwater leaching by a variety of
pollutants. A major part of the world’s food is being
supplied from fish source, so it is essential to secure the
health of fishes. In India as much as 70% of the
chemical formulations employed in agricultural
practices are believed to affect non-target organisms
and find their way to freshwater bodies, ultimately
polluting them. In the present study, one of the
organophosphorus pesticides quinalphos was used to
evaluate its short-term toxicity in freshwater fish,
Oreochromis mossambicus.
The body weights of fishes were monitored
throughout the study to ensure the effect of toxic
compounds on the general health status of the animals.
Exposure to quinalphos at the dose of 0.5µl/L for 96 h
showed a slight decrease in the body weight. However,
4
Short-term exposure to quinalphos in Oreochromis mossambicus
no significant changes in the body weights were
observed in 48 h exposed fishes. Reduction in body
weight gain may reflect a variety of responses,
including internal and external environmental factors
such as rejection of feed, treatment-induced anorexia or
systemic toxicity.
In the present study, the weights of gill and liver
were significantly decreased after 48 and 96 hours of
quinalphos treatment. The decrease in the weights of
liver might be due to the result of atrophy or necrosis of
hepatocytes (Busacker et al., 1990). Reduction in the
weights of gill could be due to degeneration of gill
filament and lamellar epithelium which was confirmed
by the histopathological study of gill in quinalphos
exposed fish (Chitra et al., 2012). In the present study
of acute toxicity testing an increased secretion of mucus
by the skin was observed which is considered as a
defensive mechanism of the fish that make its body
slippery for quick movement in the test solution and
avoids the absorption of the toxicant by the general
body surface. Similar observation was reported in the
toxic effect of two pesticides, rogor and endosulfan to
the air-breathing fish, Heteropneustes fossilis (Borah
and Yadav, 1995).
One of the early symptoms of acute pesticide
poisoning is failure of respiratory metabolism. The
respiratory potential and the oxygen consumption of the
animal are the important physiological parameters to
assess the toxic stress because it is a valuable indicator
of energy expenditure during metabolism (Proser and
Brown, 1973). It was clearly evident from the present
results that quinalphos affected respiratory rate of O.
mossambicus. In the present study, decrease in oxygen
consumption uptake from quinalphos-exposed water
may be mainly due to shrinkage of the respiratory
epithelium (Chitra et al., 2012) or possibly due to
mucus accumulation on gills.
In such a case, because of decrease in oxygen
consumption also reported to cause pronounced
haematological changes (Tilak and Satyavardhan,
2002). According to Blaxhall and Daisley (1973), the
determination of haemoglobin concentration can be a
good indicator of detecting anemic conditions in fish. In
the present study quinalphos at the dose of 0.5µl/L for
48 and 96 hours showed a significant decrease in the
level of Hb (%) and total RBC whereas WBC count
was increased significantly when compared to the
control animals and this could be due to haemodilution,
a mechanism that reduces the concentration of the
pollutants in the circulatory system (Smit et al., 1979).
The other possible reason for the decrease in the
haemoglobin concentrations of fishes under toxic stress
might be due to either an increase in the rate at which
the haemoglobin is destroyed or due to decreased rate
of haemoglobin synthesis (Reddy and Bashamohideen,
1989) or might be attributed to depression/exhaustion
of haemopoietic potential of the fish (Seth and Saxena,
2003).
J. Adv. Lab. Res. Biol.
Chitra et al
In the present study decrease in total red blood
cells (RBC) count during quinalphos treatment
suggesting a decrease in erythropoietic activity or
severe anemic state. White blood cells (WBC), or
leukocytes, are important component of the immune
system involved in defending the body against both
infectious diseases and foreign materials. The increased
total leucocytes count of the quinalphos-exposed fish in
the present study indicates increased defensive reaction
against the stressors. The increase in WBC count can be
correlated with an increase in antibody production
which helps in survival and recovery of the fish
exposed to pesticide (Joshi et al., 2002). In the present
study, the significant increase in WBC count indicates
hypersensitivity of leucocytes to quinalphos and these
changes may be due to immunological reactions to
produce antibodies to cope up with the stress induced
by quinalphos. Increased WBC count has also been
reported in fishes exposed to other certain xenobiotics
like endosulfan (Abidi and Srivastava, 1988).
In fish, proteins are the primary energy source and
are involved in regulating physiological and metabolic
processes in the body and play a vital role as energy
precursors in fishes exposed to stress conditions. In the
present study, decrease in blood protein and tissue
protein after 48 and 96 hours of quinalphos exposure
could be attributed to the enhanced activities of
proteases, lower protein synthesis at the transcriptional
level and increased proteolysis due to utilization for
metabolic processes under quinalphos toxicity. The
decrease in the protein content may also be due to its
utilization to mitigate the energy demand when the
fishes are under stress (Chandravathy and Reddy, 1994;
Soyingbe et al., 2012).
Carbohydrates are the first substrates to be utilized
in metabolism more under toxic stress conditions.
Changes in blood glucose have been suggested as
useful general indicator of environmental stress in fish
(Nemcsok and Boross, 1982). In the present study, the
significant increase of plasma glucose level might have
resulted from gluconeogenesis to provide energy for the
increased metabolic demands imposed by quinalphos
stress, particularly in osmoregulation which may
contribute to the restoration of plasma osmolarity.
In the present study exposure to quinalphos at the
dose of 0.5µl/L for 48 and 96 hours showed a
significant increase in the level of glucose in liver,
muscles and gills than that of the control group. This
could be due to increased glycogenolysis or decreased
glycogen synthesis. Depletion in the level of glucose in
gill tissue might be due to damage to the surface of gill
filaments when the freshwater fishes were exposed to
quinalphos.
5.
Conclusions
It can be concluded from the present study that
quinalphos shows high toxicity on the freshwater fish
Oreochromis mossambicus even at a short-term
5
Short-term exposure to quinalphos in Oreochromis mossambicus
exposure as the fishes are very sensitive to the presence
of even minute quantities of toxicant in their
environment causing severe metabolic stress. Thus
haematological and biochemical parameters serve as
one of the good biodetector for assessing the toxic
hazards to fish.
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